KL-6 is a high-molecular-weight sialylated glycoprotein, classified as a cluster 9 pulmonary cell antigens, and is a sensitive marker for the clinical diagnosis of interstitial pneumonia and its activity, especially in the acute phase. Additionally, it is necessary to diagnosis that patient was not pneumothirax, pulmonary thromboembolism and heart failure. In this study, we evaluated a new assay system based on chemiluminescence EIA (CLEIA) on a fully automated analyzer. Both plasma and serum samples were used, and the master calibration method was applied, eliminating the need for a standard curve preparation. The assay time was shortened to less than 1 hour. Good correlations were observed between this assay and conventional assay kits, y = 1.094x-6.849, r = 0.986 using 326 samples, and between serum and plasma y = 0.997x-1.211, r = 0.997 using 225 paired samples. In addition, the alteration of KL-6 concentration in patients undergoing chemotherapy treatment resulted in similar results that with conventional kits, and elevated KL-6 concentrations were observed in samples from patients with interstitial pneumonia. These results underscore the usefulness of this new assay kit as a rapid test, particularly for the medical examination of outpatients and the treatment of emergency cases in the acute phase of interstitial pneumonia.